The last two decades have seen a significant increase in the bacterial resistance to synthetic antibiotics placed on the market. The gram-negative bacteria of the Enterobacteriaceae family, such as Klebsiella sp. and Proteus sp., which were formerly susceptible to medicines are currently the main cause of hospital infections in several developing countries (Mendes, 2000). Consequently, an increase in the fatality-rate of immuno-depressed patients infected with these bacteria has been reported. In response to this problem, several compounds of proteic origin having anti-microbial activity have been isolated from different species of plants, mammals and microorganisms (Campos et al., 2006; Bevins, 2006).
Various plant proteins have demonstrated activity against bacteria and fungi (Selitrenikkoff, 2001). They normally have a preponderant role in the plant's defense, protecting it from environmental pathogens. These proteins have been classified within various groups and families in accordance with their structural and functional similarities. These include cyclotides, defensins, γ-thionins, Lipid-Transporting Proteins (LTPs), lectins, digestive enzyme inhibitors and various others (Selitrenikkoff, 2001, Pelegrini and Franco, 2005; Franco et al., 2002; Pelegrini et al., 2006).
Glycine rich proteins (or GRPs) consist of a new group of plant defence molecules described as having anti-microbial activity. They were initially characterised as storage proteins, being used as essential sources of amino acids (Mousavi, 2005). Previous studies have indicated the activity of glycine rich proteins in adapting to cold and in increasing tolerance to low temperatures, functioning as RNA bonding factors (Shinozuka, 2006). Furthermore, it has been demonstrated that the expression of its corresponding mRNA increases in plants exposed to low temperatures (Shinozuka, 2006). Also, it has been noted that glycine rich proteins may alter the germination and growth of plants under stress conditions, such as high salt concentrations or dehydration, (Kwak, 2005). Former studies indicated that eight glycine rich proteins isolated from seeds of Triticum kiharae did not demonstrate anti-microbial activity against bacteria but did, however, show activity against filamentous fungi, such as Helminthosporium sativum and Fusarium culmorum (Egorov, 2005). GRPs may be characterised by their high percentage of glycine residues and their primary sequence but, however, this varies with each plant and organism (Mousavi, 2005). Glycine rich proteins can be classified within three different groups according to their glycine content. The first group contains proteins presenting over 70% glycine residues in the amino acid sequence, such as GRPs of tomato and saltbush (Ringli, 2001). There is also a third group that includes proteins of high glycine content but having no specific domains (Ringli, 2001). The proteins from this group are normally hydrophobic rather than hydrophilic with this trait possibly being caused by the presence of tyrosine and phenylalanine (Ringli, 2001). The secondary structure of GRPs has not been fully studied but preliminary researches report that it may be predominantly β-sheet rich (Matsui, 1995).
Previous researches indicate that genes of glycine rich proteins are mainly found in vascular tissues, more specifically in the xylem (Ryser, 1992; Keller, 1988; Keller, 1989), despite that they have also been found in hypocotyls and pistils (Ye, 1991). The expression of GRP genes seems to be linked to inducement by stress and influenced by ambient changes, such as wounding, hormone treatment, low temperatures and dehydration (Keller, 1988; de Oliveira, 1990; Bergeron, 1994; Keister, 1995; Laberge, 1993; Condit; 1987).
This work had the objective of purifying and characterizing an anti-microbial protein from guava seeds (PgAMP1) similar to glycine rich proteins. This protein proved capable of inhibiting the growth of two gram-negative bacteria, both recognized as causing hospital infections, as well as urinary and gastrointestinal infections. This is the first report of a protein from the group of glycine rich proteins that demonstrates activity against human pathogenic bacteria.
The PgAMP1 protein identified from the present research and object of the present invention proved capable of inhibiting the growth of two gram-negative bacteria (Proteus sp. and Klebsiella sp.) and the sequence of 55 amino acids allowed its classification as a member of the glycine rich proteins.
The search for prior references disclosed documents describing the anti-bacterial activity of essential oils or extracts (aqueous, hydroalcoholic, ethanolic, methanolic and/or chloroformic) of Psidium guajava against different bacteria species (refer to Table 1, below). Different parts of the plant were used to obtain the extracts, mainly the leaves and the bark of the stem. There are no reports of the use of seeds of P. guajava for obtaining compounds having anti-bacterial activity.
TABLE 1micro-organisms and respective reports of anti-bacterial activityin extracts or essential oils of Psidium guajava.SpeciesReferencesActinomyces sp.Razak et al. (2006).Bacillus anthracisAkinpelu & Onakoya (2006).Bacillus cereusAkinpelu & Onakoya (2006); Arima & Danno(2002).Bacillus subtilisAkinpelu & Onakoya (2006); Karawya et al.(2001); Martinez et al. (1997); Rabe & vanStaden (1997); Sanches et al. (2005).ClostridiumAkinpelu & Onakoya (2006).sporogenesCorynbacteriumAkinpelu & Onakoya (2006).pyogenesEscherichia coliAbdelrahim et al. (2002); Akinpelu &Onakoya (2006); Carvalho (2002); Chan etal. (2006); Martinez et al. (1997); Rivera deLeon et al. (2001); Vieira et al. (2001);Voravuthikunchai et al. (2004).KlebsiellaAbdelrahim et al. (2002); Akinpelu &pneumoniaeOnakoya (2006); Rivera de Leon et al.(2001).MycobacteriumKarawya et al. (2001).phleiPropionibacteriumQadan et al. (2005).acnesProteus mirabilisGonçalves et al. (2005); Karawya et al.(2001).Proteus morganiiKarawya et al. (2001).Proteus spp.Carvalho (2002); Chan et al. (2006).Proteus vulgarisAbdelrahim et al. (2002); Karawya et al.(2001).PseudomonasAbdelrahim et al. (2002); Akinpelu &aeruginosaOnakoya (2006); Carvalho (2002); Chah etal. (2006); Gnan & Demello (1999);Martinez et al. (1997); Rivera de Leon et al.(2001).PseudomonasAkinpelu & Onakoya (2006).fluorescensSalmonellaArima & Danno (2002).enteritidisSalmonellaLutterodt et al. (1999).paratyphiSalmonella spp.Carvalho (2002).Salmonella typhiLutterodt et al. (1999).SalmonellaGnan & Demello (1999); Lutterodt et al.typhimurium(1999).ShigellaAkinpelu & Onakoya (2006); Ali et al.dysenteriae(1997); Ali et al. (1996); Lutterodt et al.(1999).Shigella flexneriLutterodt et al. (1999).Shigella sonneiLutterodt et al. (1999).Shigella sppCarvalho (2002); Chah et al. (2006).StaphylococcusAbdelrahim et al. (2002); Akinpelu &aureusOnakoya (2006); Betoni et al. (2006); Chahet al. (2006); Gnan & Demello (1999);Gonçalves et al. (2005); Jaiarj et al. (1999);Lutterodt et al. (1999); Martinez et al.(1997); Nascimento et al. (2000); Qadan etal. (2005); Rabe & van Staden (1997);Rivera de Leon et al. (2001); Sanches et al.(2005); Vieira et al. (2001).StaphylococcusGnan & Demello (1999); Qadan et al.epidermidis(2005); Rabe & van Staden (1997).StreptococcusAkinpelu & Onakoya (2006).faecalisStreptococcusRazak et al. (2006).mitisStreptococcusGnan & Demello (1999); Gonçalves et al.pyogenes(2005).StreptococcusRazak et al. (2006).sanguinisVibrio choleraeLutterodt et al. (1999).
The documents encountered when researching antecedents also confirmed that there is no consensus in relation to the species for which the extracts of P. guajava showed inhibitory activity. Some studies reported, for example, anti-microbial activity of P. guajava on bacteria of the genera Proteus (Abdeirahim et al., 2002; Carvalho, 2002; Chah et al., 2006; Gonçalves et al., 2005; Karawya et al., 2001) and Klebsiella (Abdelrahim et al., 2002; Akinpelu & Onakoya, 2006; Rivera de Leon et al., 2001), while others (Rabe & van Staden, 1997; Gnan & Demello, 1999; Nascimento et al., 2000) did not identify any activity on these bacteria. Therefore, determination of the composition of the extracts (according with the origin of plant material or the solvents used in the extraction, for example) proved to be an important factor when verifying the anti-bacterial activity of P. guajava. 
Some of the compounds from the extracts identified as having anti-microbial activity included flavonoids, such as guaijaverin, quercetin (Arima & Danno, 2002 and Rabe & van Staden, 1997), morin-3-O-α-L-Iyxo-pyranoside and morin-3-O-α-L-arabo-pyranoside (Arima & Danno, 2002; JP2004250406); triterpenes, such as α and β-amyrin (Sanches et al., 2005); sesquiterpenes such as caryophylene, aromadendrene, α and β-selinene or β-bisabolene (Karawya et al., 2001); sterols such as β-sitosterol (Sanches et al., 2005) and tannins (Akinpelu & Onakoya, 2006). None of these works, however, describe the peptide PgAMP1 or any other protein rich in glycine derived from P. guajava. 
The glycine rich proteins are noted for their anti-microbial activity (Egorov et al., 2005; Park et al., 2000; van Kan et al., 2001; CN1699409; CN1699416; EP1693381), but there are nevertheless few studies that relate to a specific action against bacteria (Park et al., 2000; van Kan et al., 2001; EP1693381).
Park et al. (2000) describe two new peptides isolated Capsella bursa-pastoris that present inhibitory activity against gram-negative bacteria. Van Kan et al. (2001) describe the action of clavanin A on the bacteria Micrococcus flavus. 
European patent application EP1693381 describes several sequences of glycine rich proteins that exhibit anti-bacterial activity. The peptides described in this work have molecular weights under 10 kDa and are derived from various organisms ranging from man to (Saccharomyces cerevisiæ). Due to their antibiotic activity, the inventors propose the use of these peptides in the prevention and/or treatment of infectious diseases caused by bacteria and, more specifically, in the cases where the pathogenic organisms have developed resistance to the antibiotics commonly prescribed. To confirm the anti-bacterial activity of these sequences, the inventors advanced experiments involving the peptides described and the species Escherichia coli and Bacillus subtilis. 
It should be noted that the present invention represents a technologic advance considering that despite the existence of reports about the anti-bacterial activity of glycine rich proteins, none of these documents describe a peptide that is derived from P. guajava or that shows action against the bacteria of genera Proteus sp. and Klebsiella sp.